Methods and preparations for curing critically ill patients

ABSTRACT

The present invention pertains to the use of a blood mannan-binding lectin (MBL) regulator for the manufacture of a life saving drug to treat or cure a critically ill patient. It further involves the use of measurements of MBL to predict mortality in critically ill ICU patients. One further aspect of present invention is to the use of monomers and oligomers of MBL in prophylactic and/or curative treatment of patients admitted to intensive care units (ICUs).

TECHNICAL FIELD

The present invention pertains to the use of a blood mannan-bindinglectin (MBL) regulator for the manufacture of a life saving drug totreat or cure a critically ill patient. It further claims the use ofmeasurements of MBL to predict mortality in critically ill ICU patients.One further aspect of present invention is to the use of monomers andoligomers of MBL in prophylactic and/or curative treatment of patientsadmitted to intensive care units (ICUs).

BACKGROUND OF THE INVENTION

Mortality among patients with prolonged critical illness exceeds 20percent, with most deaths being attributable to sepsis andmultiple-organ failure (Van den Berghe G et al., N Engl J Med2001;345(19):1359-67; Takala J, et al. N Engl J Med1999;341(11):785-92). Each year, these conditions affect more than 500000 patients in the United States alone (Wheeler A P et al. N. Engl. J.Med. 1999;340(3):207-14). An increased susceptibility to severeinfections during critical illness, as well as adverse effects of anexcessive systemic inflammatory response on organ function may beoperative.

The average serum concentration of MBL in the adult population isbetween 1000-2000 ng/ml with very large variations (Turner M W andHamvas R M. Rev. Immuno-genet. 2000;2(3):305-22). The between-subjectsdifferences in serum concentrations are primarily caused by geneticfactors. Point mutations within exon 1 as well as in the promoter regionof the MBL gene occur with high incidence. As a consequence,approximately one third of the population have MBL concentrations below500 ng/ml, and more than 10% have concentrations below 50 ng/ml(Steffensen R, et al. J. Immunol. Methods 2000;241(1-2):33-42).Normally, within-subject variations of MBL levels are very small (HansenT K, et al J. Clin. Endocrinol. Metab 2001;86(l1):5383-8), but serumconcentrations increase during acute phase responses (Thiel S, et al.Clin. Exp. Immunol. 1992;90(1):31-5) and can be specifically induced bygrowth hormone (GH) administration (Hansen T K, et al. J. Clin.Endocrinol. Metab 2001;86(11):5383-8). Deficiency of MBL is associatedwith an increased incidence of infections (Super M, et al. Lancet1989;2(8674):1236-9; Koch A, et al. JAMA 2001;285(10):1316-21;Summerfield J A, et al. BMJ 1997;314(7089):1229-32; Garred P, et alLancet 1995;346(8980):941-3), but due to the redundancy of the immunesystem the increased risk may only be apparent if other coexistingimmunological abnormalities are present. In line with this, it wasrecently reported that low levels of MBL in patients receiving cancerchemotherapy are associated with an increased frequency of febrileneutropenic episodes and severe infections (Neth O, Hamn I, et al.Lancet 2001;358(9282):614-8.; Peterslund N A, et al. Lancet2001;358(9282):637-8). The impact of MBL concentrations on the course ofdisease in otherwise immunocompetent critically ill patients has notbeen studied yet and is an object present invention.

By present study was investigated how MBL affects outcome in criticallyill patients. In all patients, irrespective of the treatment, the MBLconcentrations increased significantly with the time of intensive care(P<0.0001), a rise that was independent of the baseline MBLconcentration, but attributable to the survivors. By present inventionit was demonstrated that low MBL levels were at negative outcome incritically ill patients. MBL concentrations at baseline were almostthree times higher in survivors than in non-survivors (p=0.04).

This invention demonstrates that restoring MBL levels in critically illpatients to normal levels, preferable to a level above 250 μg/l serum,more preferably to a level above 500 μg/l serum, more preferably to alevel above 1000 ng/ml and most preferably between 1000 ng/ml and 2000ng/ml can be used to improve outcome such as the survival of criticallyill patients in the intensive care unit (ICU). However, after thisinvention, it will be clear for the man skilled in the art that alsoactive MBL derivatives or compounds of the group of biologically activesubstances, which stimulate late hepatocytes to synthesise and/orrelease of MBL and consequently increase the level of circulating MBLcan be used to obtain the same outcome.

The MBL is obtainable from various sources.

Mannan-binding lectin was first isolated from human serum in 1983(Kawasaki N. et al, J. Biochem (Tokyo), 1983, 94, 937-947) by affinitychromatography on mannan-Sephrarose (mannan coupled to a Sepharosematrix) in the presence of Ca-ions. Elution of MBL from the affinitycolumn was performed by means of EDTA Improved methods of manrosebinding protein purification have been described in Ezewkowitz RaymondU.S. Pat. No. 5,270,199; S. M. Tan et al Biochemical journal Vol. 319,no 2, 15 October 1996, pages 329-332; R. Koppel et al. Journal ofchromatography B.: Bio-medical Applications., vol. 662, no 2, 1994 pages191-196.; D. C. Kilpatrick Transfusion Medicine, vol. 7 , no. 4, 1997pages 289-294 and WO 99/64453 Laursen Inga. MBL can be isolated bypassage down a mannose-Sepharose column as described by Wild et al.,Biochem. J., 210: 167-174 1983 or Drickamer et al., J. of Bio.Chemistry, 261: 6878-6887 1986. MBL can be produced in engineered cells.Recombinant MBL has been produced by mammalian cell culture (Ezekowitz,U.S. Pat. No. 5,270,1999) such as in myeloma cells, Chinese hamsterovary (CHO) cells, human hepatocytes, and human embryonic kidney (HEK)cells (Vorup-Jensen,-T et al. Int-Immunopharmacol. April2001; 1(4):677-87) or by expression of MBL in methylotrophic yeast strains as forinstance described in U.S. Pat. No. 6,337,193.

Clinical grade MBL has been obtained and shown to be safe upon infusionIt has for instance already been demonstrated in patients with longdisease that pooled human donor plasma derived MBL can safely beadministered to patients (Garred,-Peter; et al Pediatr-Pulmonol. March2002; 33(3): 201-7). Moreover, present invention demonstrated for thefirst time an efficient MBL therapy for critically ill patients in theICU, which were not previously been immunocompromised (e.g. notimmunosupressed after organ transplantation or by disease).

Furthermore it will be clear for the man skilled in the art thatcompounds of the group of biologically active substances which stimulatehepatocytes to synthesise and or release MBL and consequently increasethe level of circulating MBL can be used to in a prophylactic ortherapeutic treatment to improve outcome and survivability of criticallyill patients in the ICU. Such compound with an activity of promoting thesecretion of MBL were already well disclosed before the moment of thisinvention such as growth hormone (Hansen,-T-Ket alJ-Clin-Endocrinol-Metab. November 2001; 86(11): 5383-8).

The general structure of MBL is shown in EP0375736B1 May 13, 1998 Aug.5, 1988.

Nucleic acid, for example, DNA, encoding MBL can be isolated by standardtechniques as for instance described in U.S. Pat. No. 5,270,199. Forexample, oligonucleotide probes specific for the nucleic acid may beconstructed and used to probe either genomic or cDNA libraries, asdescribed by Drickamer et al, J. Biol. Chem., 261:6878 (1986),Alternatively, gene fragments from related genes can be used as probes.Preferably, the probe is homologous to a region of the carbohydratebinding domain of MBL. The clones isolated by this technique containengineered nucleic acid. Once isolated, the gene encoding MBL is usefulfor producing recombinant MBL, or peptide fragments thereof In addition,the nucleic acid can be modified by standard techniques in order toexpress modified peptides. For instance, a human liver cDNA library hasbeen constructed by standard technique as described by Woods et al.Proc. Natl. Acad. Sci. USA. 5661, 1982. This library was probed using agel purified radiolabelled rat MBL-C cDNA sequence digested with XhoIand EcoRI as described by Drickamer (J. Biol. Chem. 263:9557, 1988).Such probe can be used under non-stringent conditions to identifyPotentially useful clones ( Kwiatkowski et al. 323 Nature 455, 1986;Messing et al. Proc. Nat. Acad. Sci. USA 74:3642, 1977). A MBL cDNAclone has for instance been used as a probe for human genomic library.Such library can for instance be constructed by standard techniques andclones, which hybridised under stringent conditions can be isolated.

Expression of MBL peptide fragments can be done by standard procedures.For example, the desired region of the MBL encoding DNA, preferably thecDNA, can be isolated from one of the above-described clones andinserted into any one of several standard expression vectors. Apreferred region for expression is that encoding the carbohydratebinding lectin isolated from the coelornic fluid of a sea urchin A.crassispina (Giga et al., J. Biol. Chem 13: 6197, 1987); a chickencartilage core proteoglycan protein (Shigaku et al., Proc. Natl. Acad.Sci. USA 83:5081, 1986) and the IgE Fc receptor (Ikuta et al., Proc.Natl. Acad. Sci. USA 84:819, 1987).

Antibodies to such expressed peptides or to MBL itself can be producedby standard techniques. They may be monoclonal or polyclonal and areuseful for identification of the peptides within animal serum or inclinical diagnostic tests.

A number of carbohydrate-binding proteins (lectins), are known in man.One group is the C-type lectins. The C-type lectins contain acalcium-dependent carbohydrate recognition domain (a C-type CRD) (Weis WI, Taylor M E and Drickamer K (1998) Immunological Reviews 163: 19-34).Mannan-binding lectin (MBL), synonymous to mannose-binding lectin,mannan-binding protein or mannose-binding protein (MBP), belongs to thesubgroup of C-type lectins, termed collecting. These soluble proteinsare composed of subunits presenting three CRDs attached to a collagenousstalk (Holmskov, U., Malhotra, R., Sim, R. B., and Jensenius, J. C.(1994) Immunol. Today 15:67-74). MBL interact with carbohydratespresented by a wide range of micro-organisms and accumulating evidenceshows that it plays an important role in the innate immune defence(Turner, M. W. (1996) Immunol. Today 17:532-540).

When bound to carbohydrate MBL is able to activate the complementsystem. The complement system may be activated via three differentpathways: the classical pathway, the alternative pathway, and themannan-binding lectin (MBL) pathway that is initiated by the binding ofMBL to carbohydrates presented by micro-organisms. The components of thealternative pathway and of the MBL pathway are parts of the innateimmune defence, also termed the natural or the non-clonal, immunedefence, while the classical pathway involves cooperation withantibodies of the specific immune defence (Janeway C A, et al (1999)Immunobiology, the immune system in health and disease, Fourth Edition,Churchill Livingstone). MBL is synthesised in the liver by hepatocytesand secreted into the blooi It binds to carbohydrate structures onbacteria, yeast, parasitic protozoa and viruses, and exhibitsantibacterial activity through killing of the micro-organisms byactivation of the terminal, lytic complement components or throughpromotion of phagocytosis (opsonization). The sertiform structure of MBLis somewhat similar to the bouquet-like structure of C1q, theimmunoglobulin-binding subcomponent of the first component in theclassical pathway (Turner, M. W. (1996) Immunol. Today 17:532-540). C1qis associated with two serine proteases, C1r and C1s, to form the C1complex. Simlarly, MBL is associated with the serine proteases MASP-1(Matsushita, M. and Fujita, T (1992). J. Exp. Med. 176:1497-1502),MASP-2 (Thiel S, et al. Nature, 386(6624): 506-510), MASP-3 (Dahl MR, etal. Immunity. 2001;15(1):127-35) and an additional protein called MAp19(Stover C M, et al J Immunol 162: 3481-3490). MASP-1, MASP-2 and MASP-3have modular structures identical to those of C1r and C1s (Dahl M R, etal Immunity. 2001;15(l):127-35). The binding of MBL to carbohydratesinduces the activation of MASP-1, MASP-2 and MASP-3. Activated MASP-2then generates the C3 convertase, C4bC2b, through cleavage of thecomplement factors C4 and C2 (Thiel Set al (1997) Nature, 386(6624):506-510). Reports suggest that MASP-1 may activate C3 directly(Matsushita M. et al J. Exp. Med. 1992; 176 (6): 1497-502). The possiblesubstrates for MASP-3 is unknown. Nothing is known about thestoichiometry and activation sequence of the MBL/MASP complexes. ML hasalso been characterised in other animals such as rodents, cattle,chicken and monkeys.

The human MBL protein is composed of up to 18 identical 32 kDapolypeptide chains (Lu, J., et al. (1990) J. Immunol. 144:2287-2294),each comprising a short N-terminal segment of 21 amino acids includingthree cysteine residues, followed by 7 repeats of the collagenous motifGly-X—Y interrupted by a Gln residues-followed by another 12 Gly-X—Yrepeats. A small 34 residue ‘neck-region’ joins the C-terminalCa²⁺-dependent lectin domain of 93 amino acids with the collagenous partof the molecule (Sastry, K., et al. (1989) J. Exp. Med. 170:1175-1189).

The collagenous regions of the polypeptide chain combines in subunitscomposed of these polypeptide chains which are covalently linked bydisulphide bridges, both between individual subunits and between thepolypeptide chains in each subunit (Turner, M. W. (1996) Immunol. Today17:532-540).

The position of these disulphide bridges has, however, not been fullyresolved. SDS-PAGE analysis under non-reducing conditions of MBL showsbands with an apparent molecular weight (m.w.) larger than 200 kDapresumably representing blocks of 3×3, 4×3, 5×3 and even 6×3 complexesof covalently assembled subunits (Lu, J., et al. (1990) J. Immunol.144:2287-2294).

The actual number of subunits in the natural human MBL protein has beencontroversial. Lipscombe et al. obtained data by use ofultracentrifugation suggesting 25% of human serum MBL to be made of 2-3trimers and only a minor fraction reaching the size of 6 trimers(Lipscombe, R. J., et al. (1995) Immunology 85:660-667). The relativequantification was carried out by densitometry of Western blotsdeveloped by chemiluminescence. By SDS-PAGE analysis of fractions fromion exchange chromatography it was found that the predominant species ofcovalently linked MBL subunit chains consisted of tetramers while onlypentameric or hexameric complexes activated complement (Lu, J., et al.(1990) J. Immunol. 144:2287-2294.). Gel permeation chromatography (GPC)analysis, in contrast, suggests that MBL is comparable in size with theC1 complex GPC can be carried out under conditions which allow for astudy of the importance of weak protein-protein interactions in theformation of MBL molecules and, in combination with standard MBL assaytechniques, also allows for unbiased determination of the MBL content inthe GPC fractions.

The concentration of MBL in human serum is largely geneticallydetermined, but reportedly increases up to threefold during acute phasereactions (Thiel S. et al. (1992) Clin Exp Immunol 90: 31-35). Threemutations causing structural alterations as well as two mutations in thepromotor region are associated with MBL deficiency (Madsen, H. O., etal. (1994) Immunogenetics 40:37-44). MBL deficiency is associated withsusceptibility to a variety of infections. Examination of five adultindividuals with unusual and severe infections showed three to behomozygous for structural MBL mutations and two to be heterozygous(Summerfield J A, et al (1995) Lancet 345: 886-889). Investigation of229 children referred to the Danish National Hospital because ofnon-HIV-related immunodeficiency showed a tenfold higher frequency ofhomozygosity for structural MBL mutant alleles than seen in a controlgroup (Garred P, et al. (1995) Lancet 346: 941-943). Allotyping of 617consecutively hospitalized children at St Mary's Hospital in Londonshowed significantly higher frequency of homozygosity and heterozygosityfor mutant allotypes in the infected children than in the noninfected(Summerfield J A, (1997) BioMed J 314: 1229-1232).

MBL can bind to a wide range of oligosaccharides. As MBL does notusually recognise self-determinants, but is well suited to interactionswith microbial cell surfaces presenting repetitive carbohydratedeterminants. In vitro, yeast (Canidida albicans and Cryptococcusneoformans), viruses ( HIV-1, HIV-2, HSV-2, and various types ofinfluenza A) and a number of bacteria have been shown to be recognisedby MBL. In the case of some bacteria, the binding with MBL is impairedby the presence of a capsule (van Emmerik, L C, et al. (1994) Clin. Exp.Immunol. 97:411-416). However, even encapsulated bacteria (Neisseriameningitidis) can show strong binding of MBL (Jack D L, et al. (1998) B.J Immunol 160: 1346-1353),

The micro-organisms, which infect MBL deficient individuals, representmany different species of bacterial, viral and fungal origin (Turner, M.W. (1996) Immunol. Today 17:532-540; Summerfield J A, et al. (1997)BioMed J 314: 1229-1232). MBL may be a general defence molecule againstmost bacteria, and thus be a reason why so many bacteria arenon-pathogenic.

While accumulating data support the notion of a protective effect of MBLthere are also observations suggesting that infections with somemicro-organisms, notably intracellular pathogens, attain a higherfrequency in MBL sufficient than in MBL deficient individuals (Garred,P, et al. (1994) Eur. J. Immunogen. 21:125-131 and Hoal-Van Helden E G,et al (1999) Pediatr Res 45:459-64; Hoal-Van Helden E G, et al. (1999)Pediatr Res 45:459-64). This is in concordance with the results of ananimal experiment, where an increased number of HSV-2 were found in theliver of mice pre injected with human MBL (Fischer, P B, et al. (1994)Scand J Immunol 39:439-445).

We investigated whether a low MBL level affects outcome innon-immunocompromised critically ill patients. Protracted criticalillness is associated with substantial metabolic derangement and a highrisk of death (Van den Berghe G. et al 2000; 143 (1): 1-13). Mortalityamong patients with prolonged critical illness exceeds 20 percent (Vanden Berghe G, et al. N Engl J Med 2001;345(19):1359-67; Takala J, et al.N Engl J Med 1999;341(11):785-92), with most deaths being attributableto sepsis and multiple-organ failure. Each year, these conditions affectmore than 500,000 patients in the United States alone (Wheeler A P andBernard G R. N. Engl. J. Med. 1999;340(3):207-14). An increasedsusceptibility to severe infections during critical illness, as well asadverse effects of an excessive systemic inflammatory response on organfunction may be operative.

Previously MBL infusion has been suggested for treating MBL deficiencyin immuno-compromised individuals. Immunocompromised is used in itsnormal meaning, i.e. an individual not being capable of evoking animmune response towards an infection MBL deficiency has often beendefined by an arbitrary level of about 50 ng/ml This level is oftenidentical with the sensitivity of various MBL test assays, and the levelhas therefore been set as the level for which substantially no MBL couldbe detected In the case of patients treated with chemotherapy a level of500 ng/ml has been suggested as defining MBL deficiency in thiscondition.

By the present invention it has been demonstrated that administratingMBL to ICU admitted patients may reduce the risk of death of subjectsthat were non-immunocompromised subjects during prolonged ICU stay. Thepatients in question may have a MBL level below 500 ng/ml serum.Clinical grade MBL is available and has been shown to be safe uponinfusion (Garred,-Peter; et al. Pediatr-Pulmonol. March 2002; 33(3):201-7). Production of recombinant MBL conceivably having a structure andan activity similar to that of native MBL has been attained (patentapplication PA 1999 00668/C5/KH).

SUMMARY OF THE INVENTION

The invention features the use of MBL, purified from natural sources orfrom material produced by recombinant technologies, or by any othersuitable MBL-producing cell line, for the treatment of individualsadmitted to ICUs. The MBL may be given before or after start of thetreatment and for any duration of time deemed suitable.

The invention in one aspect relates to treatment of individuals admittedto ICUs, critically ill patients or to treatment of individuals who areat risk of prolonged ICU admission due to procedures/treatment known tobe associated with allocations to ICUs (e.g. major surgery).

Accordingly, complications arising during ICU-stay are likely to exposethe individual in question to a higher risk of inflammatory conditionsand indeed death. It is possible according to the invention toprophylactically treat the patients before or duringprocedures/treatments (e.g. major surgery) known to be associated with arisk of prolonged ICU admission. By prophylactically treating theICU-complications before or during a treatment known to be associatedwith a risk of prolonged ICU admission it is possible to reduce themortality from sepsis and septic shock arising during the ICU-stay.

In another aspect the present invention is related to the use of acomposition comprising at least one mannan-binding lectin (MBL)polypeptide monomer, or at least one oligomer comprising at least onemannan-binding lectin (MBL) polypeptide monomer, in the manufacture of amedicament for prophylactic, ameliorating or curative treatment of acondition obtained during intensive care, in an individual initiallyhaving plasma levels of MBL below 500 ng/ml.

In yet another aspect there is provided a method to predict the risk offatal outcome in an individual allocated to ICUs through measurements ofthe concentration of MBL in plasma or serum obtained from the individualand estimation of the probability on the basis of the measuredconcentration.

DEFINITIONS

The term “systemic inflamatory response syndrome (SIRS)”, as used hereinrefers to the uncontrolled disease process which ensues an initialinsult and which gives rise to a multi system disturbance secondary toinflammatory mediators released during shock. It can mean a response toan inflammation or injury that can be infections or non-infectious,defined by having two of the following: 1) Temperature above 38 degreesC. or less than 36 degrees C., 2) Heat rate >90, 3) Respiration rate >20or Paco2<32 torr, 4) WBC>12,000/mm3 or <40000, or >10% bands.

The term “Sepsis”, as used herein refers to “SIRS”, as described above,which is particularly caused by an infectious insult leading to theinitial shock phase.

The term “Bacteraemia”, as used herein means the presence of bacteria inthe blood-stream detected by blood cultures.

The term “Septic shock” as used herein means Sepsis with a systolic BP<90 mm or drop of 40 mm Hg from baseline value in absence of othercauses. Severe sepsis may cause organ dysfunction.

The term “Critical ill patients” (CIP) as used herein means patientswhich have sustained or are at risk of sustaining acutelylife-threatening single or multiple organ system failure due to diseaseor injury, a patient who is being operated and where complicationssupervene, and a patient who has been operated in a vital organ withinthe last week subject to major surgery within the last week. Usually andpreferably, these conditions necessitate prolonged minute to minutetherapy an/or observation, usually and preferably in an intensive careunit (ICU) which is a part of an hospital or alike capable of providinga high level of intensive therapy in terms of quality and immediacy.Critical illness might be explained as a disease or state in patients inwhich death is possible or imminent and whereby the patients aremaintained under total or partial parenteral nutrition in an intensivecare unit (ICU), a hospital facility or alike for provision of intensivenursing and medical care, characterised by high quality and quantity ofcontinuous monitoring. In a more restricted sense, the term a“critically ill patient”, as used herein refers to a patient who hassustained or are at risk of sustaining acutely life-threatening singleor multiple organ failure due to disease or injury, or a patient who isbeing operated and where complications supervene. In a even morerestricted sense, the term a “critically ill patient”, as used hereinrefers to a patient who has a sustained or are at risk of sustainingacutely life-threatening single or multiple organ system failure due todisease or injury. Similarly, this definition applies to similarexpressions such as ‘critical illness in a patient’ and a ‘patient iscritical ill’. Examples of a critically ill patient is a patient in needof cardiac surgery, cerebral surgery, thoracic surgery, abdominalsurgery, vascular surgery, or transplantation, or a patient sufferingfrom neurological diseases, cerebral trauma, respiratory insufficiency,abdominal peritonitis, multiple trauma, severe burns, or criticalillness polyneuropathy.

DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENT

Indication

Previously MBL infusion has been suggested for treating MBL deficiencyin imuno-compromised individuals. Immunocompromised is used in itsnormal meaning, i.e. an individual not being capable of evoking animmune response towards an infection. MBL deficiency has often beendefined by an arbitrary level of about 50 ng/ml. This level is oftenidentical with the sensitivity of various MBL test assays, and the levelhas therefore been set as the level for which substantially no MBL couldbe detected. In the case of patients treated with chemotherapy a levelof 500 ng/ml has been suggested as defining MBL deficiency in thiscondition

By the present invention administrating MBL to ICU admitted patients mayreduce the risk of death from sepsis and septic shock during prolongedICU stay. The patients in question may have a MBL level below 500 ng/mlserum.

Also the treatment of ICU admitted individuals may be conducted byadministering MBL to these individuals in combination with relevantantibiotics, anti-viral agents or anti-fungal agents.

In particular, individuals at risk of prolonged ICU admission due to asurgical or medical treatment will benefit from prophylactic treatmentwith MBL before, during and possibly also after the treatment in orderto reduce the mortality from complications (sepsis, septic shock ormultiple organ deficiency ) arising during the ICU-stay.

Generally all individuals being allocated to ICUs having a MBL levelbelow 500 ng/ml should be treated with MBL to reduce the risk of deathfrom complications (sepsis and septic shock) arising during theICU-stay, independent of their specific MBL level. Consequently, inparticular individuals having a MBL level below 400 ng/ml will benefit,such as individuals having a MBL level below 300 ng/ml, such asindividuals having a MBL level below 200 ng/ml, such as individualshaving a MBL level below 100 ng/ml, such as individuals having a MBLlevel below 50 ng/mL

Thus, the present invention in particular relates to the use of MBLpolypeptides or blood MBL increasing factors for manufacturing of amedicament for the treatment of individuals having a MBL level in serumin the range of 0-500 ng/ml. The condition may be due to the treatmentsat ICUs. However, the condition may also be due to a surgical or medicaltreatment known to be associated with a risk of prolonged ICU admission.

The MBL deficiency may be without known genesis, either acquired orinherited. Individuals, having a MBL level below 500 ng/ml will benefitfrom MBL treatment in general in order to prevent ICU inducedconditions.

According to one embodiment, the present invention relates to the use ofMBL or blood MBL stimulators for the manufacture of a life saving drugto treat or cure a critically ill patient.

According to another embodiment, the present invention relates to a useof MBL or blood MBL stimulators for the manufacture of a medicament totreat or cure a critically ill patient.

According to yet another embodiment, the present invention relates tothe use of MBL or blood MBL stimulators for the manufacture of amedicament to prevent that a patient becomes critical ill.

According to a further embodiment, the present invention relates to ause of MBL or blood MBL stimulators for the manufacture of a medicamentto increase the survival rate of a critically ill patient.

According to a further embodiment, the present invention relates to ause of MBL or blood MBL stimulators for the manufacture of a medicamentto reducing the time a critically ill patient ill patient stays within ahospital, for example within an ICU.

According to a further embodiment, the present invention relates to ause of MBL or blood MBL stimulators for the manufacture of a medicamentto prevent treat or cure sepsis and/or its mediators, especially in acritically ill patient.

According to a further embodiment, the present invention relates to ause of MBL or blood MBL stimulators for the manufacture of a medicamentto reduce mortality, hospitality stay, bacteraemia, need for ventilatorysupport, need for dialysis.

In a further embodiment of present invention, the critically ill patientis a patient in need of cardiac surgery.

In a further embodiment of present invention, the critically ill patientis a patient in need of cerebral surgery.

In a further embodiment of present invention, the critically ill patientis a patient in need of thoracic surgery.

In a further embodiment of present invention, the critically ill patientis a patient in need of abdominal surgery,

In a further embodiment of present invention, the critically ill patientis a patient in need of vascular surgery.

In a further embodiment of present invention, the critically ill patientis a patient in need of transplantation

In a further embodiment of present invention, the critically ill patientis a patient suffering from neurological diseases.

In a further embodiment of present invention, the critically ill patientis a patient suffering from cerebral trauma.

In a further embodiment of present invention, the critically ill patientis a patient suffering from respiratory insufficiency.

In a further embodiment of present invention, the critically ill patientis a patient suffering from abdominal peritonitis.

In a further embodiment of present invention, the critically ill patientis a patient suffering from multiple trauma.

In a further embodiment of present invention, the critically ill patientis a patient suffering from severe burns.

In a further embodiment of present invention, the critically ill patientis a patient suffering from critical illness neuropathy.

In a further embodiment of present invention, the critically ill patientis a patient being mechanically ventilated.

Furthermore, the present invention relates to a method of selling asubstance or a composition of said substance, which stimulate the levelsof circulating MBL in a subject by giving information about their novelutility, novel activity and/or novel pharmaceutical indicationsdescribed herein. One method of selling such blood MBL regulator couldbe by telling a person, for example a physician, that MBL or a factorwhich stimulate hepatocytes to synthesise and/or release of MBL andconsequently increase the level of circulating MBL maybe used to treatcritically ill patients or to reduce the mortality from complications(sepsis and septic shock) arising during the ICU-stay. Alternatively, amethod of selling a blood MBL regulators or MBL could be by distributingthe above advertising and information, whereby the media are brochures,packaging material which is used for the customer package, any printedmaterial/leaflet supplied with the drug, or patient information, labels,web sites, movies, advertising movies, videos, and the like.

Another method of selling a blood MBL regulators or MBL which is coveredby the present claims is to support a speaker giving information aboutthe novel utility, indication, and action of the blood glucose regulatoraccording to the present invention or to support an author writing anarticle giving information about the novel utility, indication, andaction of the a blood MBL regulators or MBL according to the presentinvention. Other variations hereof will be obvious for the skilled artworker, for example distributing and advertisement as the above.

MBL

The MBL composition used to manufacture a MBL medicament may be producedfrom any MBL source available. The MBL source may be natural MBL,whereby the MBL polypeptides are produced in a native host organism,meaning that MBL is produced by a cell normally expressing MBL. Oneusual method of producing a MBL composition is by extraction of MBL fromhuman body liquids, such as serum or plasma.

In another aspect the MBL polypeptide oligomers are produced by a hostorganism not natively expressing a MBL polypeptide, such as byrecombinant technology.

In a first embodiment the MBL source may be serum, from which an MBLcomposition is obtained by purifying serum, plasma, milk product,colostrum or the like by a suitable purification method, such asaffinity chromatography using carbohydrate-derivatised matrices, such asmannose or mannan matrices. Such a method is discussed in WO99/64453which is hereby incorporated by reference.

The MBL composition used to manufacture a MBL medicament preferablycomprises MBL oligomers having a size distribution substantiallyidentical to the size distribution of MBL in serum, such as a sizedistribution profile at least 80% identical to the size distributionprofile of MBL in serum, more preferred a size distribution profile atleast 90% identical to the size distribution profile of MBL in serummore preferred a size distribution profile at least 95% identical to thesize distribution profile of MBL in serum.

The matrix may be derivatized with any carbohydrate or carbohydratemixture whereto MBL binds. The matrix is preferably a mannose-, afucose, a N-acetyl-glucosamin or a glucose derivatized matrix, such asmost preferably a mannose matrix.

The selectivity of the carbohydrate-derivatized matrix is obtained bysecuring that the matrix as such, i.e the un-derivatized matrix hassubstantially no affinity to MBL polypeptides. This may be ensured whenthe matrix as such is carbohydrate-free.

The matrix may be in any form suitable for the chromatography, mostly inthe form of beads, such as plastic beads.

After application of the MBL source the column is washed, preferably byusing non-denaturing buffers, having a composition, pH and ionicstrength resulting in elimination of or lowering of the amount ofcontaminating proteins, without eluting the MBL. Such as buffer may beTBS (10 mM Tris, 145 mM NaCl, pH 7.4) with calcium ions added. Elutionof MBL is performed with a selective desorbing agent, capable ofefficient elution of MBL, such as TBS, with added EDTA, and MBLoligomers are collected. Such a purification method is described inInternational patent application (WO0070043 and WO9937676).

In a preferred aspect a clinical grade MBL composition is obtained byusing an MBL source produced by recombinant technology, wherein the MBLsource is the culture media from culturing of MBL producing cells.

Thus, the present invention encompasses MBL produced by a process ofproducing a human recombinant mannan binding lectin (MBL) polypeptide,comprising the steps of:

-   -   preparing a gene expression construct comprising a DNA sequence        encoding a human MBL polypeptide or a functional equivalent        thereof    -   transforming a host cell culture with the construct,    -   cultivating the host cell culture, thereby obtaining expression        and secretion of the polypeptide into the culture medium,        followed by    -   obtaining a culture medium comprising human recombinant MBL        polypeptides.

The culture medium comprising the human recombinant MBL polypeptides maythen be purified as described above.

The gene expression construct may be produced by conventional methodsknown to the skilled person, such as described in U.S. Pat. No.5,270,199.

In another embodiment the gene expression construct is prepared asdescribed in Danish Patent application No: PA 1999 00668 or inInternational patent application (WO0070043) having the title“Recombinant Human Mannan Binding Lectin”).

The expression is preferably carried out in e.g. mammalian cells, thepreparation according to the invention results from the use of anexpression vector comprising intron sequence(s) form an MBL gene and atleast one exon sequence. Regarding the transgenic animals as expressionsystem this term is in this context animals which have been geneticallymodified to contain and express the human MBL gene or fragments ormimics hereof.

In addition to the purification method it is preferred that the geneexpression construct and the host cell also favours production of higheroligomers, which has been found to be possible by using a geneexpression construct comprising at least one intron sequence from thehuman MBL gene or a functional equivalent thereof.

In particular the MBL composition is used for treatment and/orprophylaxis of sepsis, septic shock or multiple organ failure whichoccurs in patients treated within the intensive care unit (ICA). Suchpatients may have post-surgical critical illness, post-traumaticcritical illness or patients on ventilator in the ICU.

It is an object of present invention to use the MBL composition in alife saving treatment of critically ill patients.

A still further object of present invention is to use the MBLcomposition to reduce the time a critically ill patients stays within aICU.

Another object of present invention is to use the MBL composition tosuppress states of sepsis, septic shock or multiple organ failure.

Another object of present invention is to use the MBL composition toreduce the risk or likelihood from multiple organ failure with a provenseptic focus on post-mortem examination in a critically ill patient.

Another object of present invention is to use the MBL composition toreduce the mortality, for example, in hospital mortality, in acritically ill patient.

Another object of the present invention is to use the MBL composition toreduce the likelihood of organ replacement therapy and/or organ failure(for instance renal) in a critically ill patient.

Another object of present invention is to use the MBL composition toreduce the likelihood of hyperbillirubinemia in a critically illpatient.

Another object of present invention is to use the MBL composition toreduce the liklihood for blood stream infections in a critically illpatient.

Another object of present invention is to use the MBL composition toreduce the likelihood of disturbance in markers of inflammations and/orinflammatory response in a critically ill patient.

Another object of present invention is to use the MBL composition toreduce the use of antibiotics in a critically ill patient.

Another object of present invention is to use the MBL composition toreduce the likelihood of a critically ill patient having repetitivepositive EMGs.

Another object of present invention is to use the MBL composition toreduce the amount of red cell transfusion in a critically ill patient.

Another object of present invention is to use the MBL composition toreduce the need for invasive treatment in a critically ill patient.

Compositions

The medicament containing MBL may be produced by using the eluantobtained from the affinity chromatography as such It is howeverpreferred that the eluant is subjected to further purification stepsbefore being used.

In addition to the MBL polypeptide oligomers, the medicament maycomprise a pharmaceutically acceptable carrier substance and/orvehicles. In particular, a stabilising agent may be added to stabilisethe MBL proteins. The stabilising agent may be a sugar alcohol,saccharides, proteins and/or aminoacids. An example of a stabilisingagent may be albumin.

Other conventional additives may be added to the medicament depending onadministration form for example. In one embodiment the medicament is ina form suitable for injections. Conventional carrier substances, such asisotonic saline, may be used.

In another embodiment the medicament is in a form suitable for pulmnonaladministration, such as in the form of a powder for inhalation or cremeor fluid for topical application.

The route of administration may be any suitable route, such asintravenously, intramuscularly, subcutanously or intradermally. Also,pulmonal or topical administration is envisaged by the presentinvention.

In particular the MBL composition may be administered to prevent and/ortreat “ICU complications” in patients having clinical symptomsassociated with congenital or acquired MBL deficiency or being at riskof developing such symptoms.

The MBL composition may also be administered simultaneously,sequentially or separately with another treatment.

The MBL composition is administered in suitable dosages, in particularlyit is administered at ICU admission and maintained once, twice or thricea week at least during a part of the stay at ICU, preferably during thewhole ICU period.

Normally from 1-100 mg is administered per dosage, such as from 2-10 mg,mostly from 5-10 mg per dosage. For other indications the dosage regimemay vary.

The use of a MBL composition may also be in a kit-of-parts furthercomprising an anti-fungal, anti-yeast, anti-bacterial and/or anti-viralmedicament. The anti-viral medicament may be a medicament capable ofvirus attenuation and/or elimination.

The invention also relates to an aspect of using a measurement of theMBL level as a prognostic marker for the risk of the individual ofacquiring ICU complications and thereby an indicative of the need fortreatment. In particular a MBL level below 500 ng/ml is a prognosticmarker indicative for treatment with MBL.

Thus, the present invention also relates to a method of using a MBLpolypeptide composition for preventing and/or reducing “ICUcomplications” in an individual, the method comprising the steps of:

-   -   i) determining serum levels of MBL polypeptide in an individual,    -   ii) estimating the probability of the occurrence of ICU        complications in the individual, and optionally, administering a        MBL polypeptide composition to the individual.

The MBL level is measured in serum or plasma, and may be determined bytime resolved immunofluorescent assay (TRIFMA), ELISA, RIA ornephelometry.

Also the MBL levels may be inferred from analysis of genotypes of theMBL genes.

The invention has now been explained and accounted for in variousaspects, but additionally it will be illustrated below by FIGS. 1 and 2and table 1 and the non-limiting examples of preferred embodiments.

FIGURE LEGENDS

FIG. 1:

Serial measurements of mannan-binding lectin (MBL) concentrations inpatients receiving prolonged (>5 days) intensive care. Bars representsmedians, boxes IQRs and whiskers the 10^(th) and 90^(th) percentiles. Pvalue refers to Friedman's test for several related samples

FIG. 2:

Relative change in MBL concentrations from day 1 (Δ%) in patientsreceiving prolonged intensive care.

EXAMPLE

The following example demonstrates the results of an examination of theinfluence of MBL deficiency on outcome for individuals admitted tointensive care units.

Study Population

The study encompasses examination of 243 patients on mechanicalventilation admitted to the Department of Intensive Care Medicine,University of Leuven, Leuven, Belgium. All patients included in thisstudy received intensive care for more than 5 days.

Blood stream infection was defined by a blinded investigator as thepresence of bacterial pathogens, excluding contamination according toStrict criteria (Weinstein M P et al Clin. Infect. Dis. 1997; 24 (4):584-602.), in blood cultures obtained when central body temperaturesteeply rose above 38.5° C. The use of antibiotics was recorded as thetotal number of days on any systemic antibiotic treatment. The number ofdays during which leukopenia (≦4000 cells/μl) or leukocytosis (≧12000cells/μl) was present, and the number of days during which an episode ofhypothermia (≦36° C.) or hyperthermia (≧38° C.) occurred, were alsoanalysed. The incidence of acute renal failure requiring renalreplacement therapy was recorded. Weekly EMG screenings were performedfor the diagnosis of critical illness polyneuropathy. The cause of deathfor all patients who died was established clinically by the attendingphysician and confirmed on post-mortem examination by a pathologist whowas unaware of treatment assignment.

MBL Measurements

Blood samples were drawn within 24 h after admission to the ICU(baseline), and subsequently on days 5 and 15 and/or the last day ofintensive care (ie. the day of discharge or death) for determination ofserum MBL.

Serum MBL concentrations were measured using time-resolvedimmunofluorometric assay (IRMA) (Thiel S et al, Immunology 2002; 204).Microtiter wells (fluoroNunc, Nunc, Denmark) were coated with mannanfollowed by incubation with diluted test samples and standards.

After washing, europium labelled monoclonal anti-MBL antibody (131-1,Immuno-lex, Denmark, labelled with europium using reagents from WallacOy, Finland) was added, and after incubation and washing fluorescenceenhancement solution was added (Wallac) and the plates were read on atime resolved fluorometre (Delfia 1232, Wallac). The calibration curvewas made using dilutions of one plasma, which was kept alliquoted at−80° C. The concentration of MBL in this plasma (3.6 μg/ml) wasdetermined by comparison with highly purified MBL, which was quantifiedby quantitative amino acid analysis.

An alternative TRIFMA for analysing the MBL is coating Microtitre wells(fluoro-Nunc, Nunc, Karnstrup, Denmark) with antibody by incubationovernight at room temperature with 500 ng anti-humanMBL antibody (Mab131-1, Statens Serum Institut, Copenhagen, Denmark) in I 00 pfPBS (0.14M NaCl, 10 mM phosphate, pH 7.4). After wash with Tween-containingbuffer (TBS, 0.14 M NaCl, 10 mM Tris/HCI, 7.5 mM NaN3, pH 7.4 with 0.05%Tween20) test samples (plasma 1/20) and calibrator dilutions can beadded in TBS/Tween with extra NaCl to 0.5 M and 10 mM EDTA.

After overnight incubation at 40C and wash, the developingeuropium-labelled anti-body (12.5 ng Mab 131-1 labelled with theEu-containing chelate, isothiocyanato-benzoyl-diethylene-trianine-tetraacetic acid, according to the manufacturer, Wallac,Turku, Finland) canbe added in TBS/Tween with 25 pM EDTA.

Following incubation for 2 h and wash, fluorescence enhancement solutionis added (Wallac) and the plates are read on a time resolved fluorometre(Delfia 1232, Wal-lac). The calibration curve is made using dilutions ofone plasma, which is kept al-liquoted at −80 C.

Collected blood for TRIFMA analysis is drawn into evacuated glass tubescontaining EDTA (final concentration about 10 mM). The plasma isaliquoted and kept at −80° C. until assay. Plasma samples are similarlyobtained from healthy blood donors. The patient are free of infectionsat the time of blood sampling.

Statistical Analysis

Changes in MBL concentrations over time during the ICU-stay wereanalysed by Friedman's test. Spearmian correlation with two-tailedprobability values was used to estimate the strength of associationbetween variables. The impact of baseline MBL level on outcome variables(mortality, acute renal failure, bacteremia, prolonged need forantibiotic treatment, polyneuropathy) was assessed by multivariatelogistic regression analysis. In addition, multivariate logisticregression analysis was used to assess whether the changes in MBL overtime explained clinical outcome variables. Data are given as medianswith interquartile ranges unless specified otherwise and statisticalsignificance was assumed for P<0.05. All statistical calculations wereperformed with Statview 5.0.1. for Macintosh (SAS Institute Inc., NorthCarolina, USA).

Results

Clinical Patient Characteristics and Outcome

Of the 243 patients included in the study 49 died during intensive care.The cause of death was multiple-organ failure with or without a provenseptic focus, acute cardiovascular collapse and severe brain damage.

Bacteraemia occurred in 25% of the patients. The patients receivedtreatment with antibiotics for a median duration of 12 (IQR 6-21) days,leucopenia or leucocytosis was present for a median of 6 (IQR 2-13) daysand hypo- or hyperthermia for a median 10 (IQR 5-16) days.

Serum MBL Concentrations

Upon ICU admission, the average serum MBL concentration was 820 [IQR241-1518] μg/l which is comparable with the level documented in healthyDanish and British subjects. The number of patients with a baseline MBLlevel below 500 ng/ml and below 50 ng/ml was 40% and 8.9%, respectively.

Analysis of the treated patients, non-survivors revealed significantlylower baseline MBL concentrations as compared with survivors (387 [IQR190-1289]) μg/l vs. 897 [IQR 246-1686] μg/l, respectively; P=0.04, Table1). The fraction of patients with MBL concentrations below 500 ng/ml was54% among non-survivors as compared to 36% among survivors (P=0.02). Thefraction of patients with MBL concentrations below 250 ng/ml was 34%among non-survivors as compared to 25% among survivors, and the fractionof patients with MBL concentrations below 50 ng/ml was 14% amongnon-survivors as compared to 7% among survivors.

The MBL concentrations increased significantly with time in intensivecare (P<0.0001, FIG. 1). This rise was independent of the baseline MBLconcentration and mostly attributable to the survivors. Patients whodeveloped bacteremia revealed a lower relative increase in MBL levels onday 15 compared to those who did not develop bacteremia (P≦0.02).

Discussion

Protracted critical illness is associated with substantial metabolic andimmunological derangement and a high risk of death (Van den Berghe G. etal 2000; 143 (1): 1-13; Van den Berghe G, et al N Engl J Med2001;345(19):1359-67; Takala J, et al. N Engl J Med 1999;341(11):785-92)). We observed that low on-admission concentrations of MBL may predict apoor outcome among patients treated with intensive care.

More than two-thirds of patients admitted to intensive care unitsdevelop signs of the systemic inflammatory response syndrome (SIRS)(Brun-Buisson C. Intensive Care Med 2000; 26: S64-S74 ), either causedby infection or tissue damage, and a substantial number of thesepatients progress to shock and multiple-organ failure.

A number of publications have reported a possible association betweenlow levels of MBL and increased risk of infections particularly inpatients who are immunocompromised, such as children with immatureantibody repertoire (Koch,-A et al. JAMA Mar. 14, 2001; 285(10):1316-21), patients with AIDS (Kelly,-P. et al Gastroenterology. November2000; 119(5): 1236-42.Gastroenterology), or patients with malignanciesreceiving chemotherapy or stem cell transplantation (Neth,-O; et alLancet. Aug. 25, 2001; 358 (9282): 614-8; Peterslund,-N-A; Koch,-C;Jensenius,-J-C; Thiel,-S Lancet. Aug. 25, 2001 ; 358(9282): 637-8;Mullighan,-Charles-G; et al. Blood May 15, 2002; 99(10): 3524-9.).

In present invention the current analysis of the 243 ICU treatedpatients that were not previously immunocompremised revealed that MBLlevels on admission were almost three times higher in survivors than innon-survivors, in favour of a vulnerability associated with low MBLlevels in critically ill patients who are immunocompetent. Theassociation between low levels of MBL and outcome of treated ICUpatients was not restricted to severe MBL deficiency but was evidenteven when using a concentration of <500 ng/ml as a cut-off level forfunctional MBL deficiency as previously suggested by Peterslund et al(Lancet. Aug. 25, 2001; 358(9282): 637-8).

In conclusion low levels of MBL may predict a poor outcome in protractedcritical illness. TABLE 1 Serial measurements of MBL concentrations inpatients undergoing prolonged intensive care treatment. Died in ICU ICUsurvivor All patients n = 49 n = 194 P MBL concentrations Day 1 387(190-1287)  897 (246-1686) 0.045 (μg/l, median Day 5 460 (158-2140) 1321(346-2706) 0.012 [IQR]) Day 15 1855 (181-2594)  1934 (322-3574) 0.376Last Day 990 (240-2408) 1960 (569-3848) 0.002

1-50. (canceled)
 51. A method comprising a use of mannan-binding lectin(MBL) for the treatment of a patient suffering from systemicinflammatory response syndrome (SIRS) and/or a patient in need oftransplantation and/or for reducing the risk in a patient of acquiringSIRS.
 52. The method according to claim 51, wherein the patient is inneed of a transplantation.
 53. The method according to claim 51, toprevent the patient from acquiring SIRS.
 54. The method according toclaim 51, wherein the patient is critically ill, wherein a criticallyill patient is a patient which has sustained or are at risk ofsustaining acutely life-threatening single or multiple organ systemfailure due to disease or injury, a patient who is being operated andwhere complications supervene, and a patient who has been operated in avital organ within the last week or subject to major surgery within thelast week.
 55. The method according to claim 51, wherein said MBLtreatment is administered to increase the survival rate in the intensivecare unit (ICU).
 56. The method according to claim 51, wherein said MBLtreatment is administered to reduce the time that a patient stays withinthe hospital, for example within the intense care unit.
 57. The methodaccording to claim 54, wherein the patient suffers from post-surgicalcritical illness.
 58. The method according to claim 54, wherein thepatient suffers from post-traumatic critical illness.
 59. The methodaccording to claim 51, wherein the MBL polypeptide monomer is amammalian polypeptide monomer.
 60. The method according to claim 59,wherein said mammalian MBL polypeptide monomer is a human polypeptidemonomer.
 61. The method according to claim 51, wherein said MBLcomprises oligomers, wherein said oligomers are preferably selected fromthe group of oligomers consisting of trimers, tetramers, pentamersand/or hexamers.
 62. The method according to claim 51, wherein the MBLcomprises at least one mannan-binding lectin (MBL) polypeptide monomer,or at least one mannan-binding lectin (MBL) polypeptide oligomercomprising or at least one mannan-binding lectin (MBL) polypeptidemonomer.
 63. The method according to claim 51, wherein MBL comprises atleast one mannan-binding lectin (MBL) polypeptide oligomer comprising atleast one mannan-binding lectin (MBL) polypeptide monomer.
 64. Themethod according to claim 51, wherein the medicament is used in such waythat the blood MBL level in the patient is kept above 250 ng/ml.
 65. Themethod according to claim 51, wherein the medicament is used in such waythat the blood MBL level in the patient is kept above 500 ng/ml.
 66. Themethod according to claim 51, wherein the medicament is used in such waythat the blood MBL level in the patient is kept above 1000 ng/ml. 67.The method according to claim 51, wherein the medicament is used in suchway that the blood MBL level in the patient is kept between 1000 ng/mland 2000 ng/ml.
 68. The method according to claim 51, wherein themedicament is administered to the individual prior to another treatmentat ICUs.
 69. The method according to claim 51, wherein the medicament isadministered to the individual simultaneously, sequentially orseparately with another treatment.
 70. The method according to claim 51,wherein the medicament is administered to the individual prior to duringor after said other treatment.
 71. The method according to claim 51,wherein the patient has MBL polypeptide serum levels below apredetermined minimum MBL polypeptide serum level, wherein thepredetermined level is a concentration of 500 ng/ml.
 72. The methodaccording to claim 51, wherein said treatment is to reduce the risk of afatal outcome during intensive care treatment of an individual.